How terminal Schwann cells (tSCs), glia that cover the terminal branches of motoneurons at the neuromuscular junction (NMJ), function in synaptic maintenance and turnover remains incompletely understood. A variety of observations suggest that defects in axonal-glial interactions at the synapse and along the projecting axons lead to degenerative disease. The present project proposes a study of the role of tSCs in the normal, ongoing turnover of synaptic contacts at NMJs. While most of the area in which the motoneuron terminal contacts muscle fibers is stable in young adult mice, the site where the nerve enters the NMJ is less so. Here, contact sites are commonly lost; and this loss is associated with glial changes, including the myelination of terminal branches that were previously synaptic. This kind of loss appears to be a progressive phenomenon, becoming more prevalent with time and quite prevalent in aged animals. We have generated transgenic mice whose expression of fluorescent proteins allows the repeated, vital imaging of the cellular components of the synapse and have shown loss of synaptic sites and some of their associated glial changes. We have shown that individual tSCs partition their coverage of the synaptic site among themselves. However, we have been unable to visualize individual glial cells in a vital fashion. We have now generated mice that express a photoswitchable fluorescent protein in their SCs. This allows the visualization, marking and examination of individual cells. We will (1) utilize these mice to determine how individual tSCs participate in synaptic changes. (2) We will use electron microscopy to examine the portions of individual synapses in the process of change to determine what the light microscopic features mean at the ultrastructural level and to examine the possibility that the glia are stripping synapses from the muscle fiber. We will also determine when individual tSCs in the junction begin to express certain markers of altered glial differentiation and how these alterations relate to ongoing synaptic change. (3) Finally we will examine neuregulin signaling believed to lead to differentiation of SCs to a myelinating phenotype. We will examine the distribution of this signaling molecule in motor nerve terminals and determine how overexpression of this signaling molecule alters the number and behavior of synaptic glia and leads to structural changes in the synapse. The results of the proposed work should expand our understanding of the interactions of glia and neurons at synapses. PUBLIC HEALTH RELEVANCE: Existing and new genetically modified mice will be used to explore, via vital imaging, the function of glia at a simple, model synapse, the neuromuscular junction. The proposed research will investigate the hypothesis that glial cells initiate synaptic remodeling that occurs normally in young adults but becomes pronounced in aging. The proposed work also has relevance to dysmyelinating diseases that ultimately lead to degeneration of peripheral motor neurons.